System and method for analyte measurement using AC phase angle measurements

ABSTRACT

A method of measuring an analyte in a biological fluid comprises applying an excitation signal having a DC component and an AC component. The AC and DC responses are measured; a corrected DC response is determined using the AC response; and a concentration of the analyte is determined based upon the corrected DC response. Other methods and devices are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 10/264,890, Oct. 4, 2002, which is a divisionalapplication of U.S. patent application Ser. No. 09/530,171, filed Apr.24, 2000, which is the U.S. national stage of International PatentApplication Serial No. PCT/US98/27203, filed Dec. 21, 1998, which is acontinuation-in-part of U.S. patent application Ser. No. 08/996,280,filed Dec. 22, 1997 (now abandoned), and claims priority benefit fromeach of these applications. This application also claims the benefit ofU.S. Provisional Application No. 60/480,298, filed Jun. 20, 2003. Thecontents of each of these applications are hereby incorporated byreference herein.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to a measurement method andapparatus for use in measuring concentrations of an analyte in a fluid.The invention relates more particularly, but not exclusively, to amethod and apparatus which may be used for measuring the concentrationof glucose in blood.

BACKGROUND OF THE INVENTION

[0003] Measuring the concentration of substances, particularly in thepresence of other, confounding substances, is important in many fields,and especially in medical diagnosis. For example, the measurement ofglucose in body fluids, such as blood, is crucial to the effectivetreatment of diabetes.

[0004] Diabetic therapy typically involves two types of insulintreatment: basal, and meal-time. Basal insulin refers to continuous,e.g. time-released insulin, often taken before bed. Meal-time insulintreatment provides additional doses of faster acting insulin to regulatefluctuations in blood glucose caused by a variety of factors, includingthe metabolization of sugars and carbohydrates. Proper regulation ofblood glucose fluctuations requires accurate measurement of theconcentration of glucose in the blood. Failure to do so can produceextreme complications, including blindness and loss of circulation inthe extremities, which can ultimately deprive the diabetic of use of hisor her fingers, hands, feet, etc.

[0005] Multiple methods are known for measuring the concentration ofanalytes in a blood sample, such as, for example, glucose. Such methodstypically fall into one of two categories: optical methods andelectrochemical methods. Optical methods generally involve reflectanceor absorbance spectroscopy to observe the spectrum shift in a reagent.Such shifts are caused by a chemical reaction that produces a colorchange indicative of the concentration of the analyte. Electrochemicalmethods generally involve, alternatively, amperometric or coulometricresponses indicative of the concentration of the analyte. See, forexample, U.S. Pat. No. 4,233,029 to Columbus, U.S. Pat. No. 4,225,410 toPace, U.S. Pat. No. 4,323,536 to Columbus, U.S. Pat. No. 4,008,448 toMuggli, U.S. Pat. No. 4,654,197 to Lilja et al., U.S. Pat. No. 5,108,564to Szuminsky et al., U.S. Pat. No. 5,120,420 to Nankai et al., U.S. Pat.No. 5,128,015 to Szuminsky et al., U.S. Pat. No. 5,243,516 to White,U.S. Pat. No. 5,437,999 to Diebold et al., U.S. Pat. No. 5,288,636 toPollmann et al., U.S. Pat. No. 5,628,890 to Carter et al., U.S. Pat. No.5,682,884 to Hill et al., U.S. Pat. No. 5,727,548 to Hill et al., U.S.Pat. No. 5,997,817 to Crismore et al., U.S. Pat. No. 6,004,441 toFujiwara et al., U.S. Pat. No. 4,919,770 to Priedel, et al., and U.S.Pat. No. 6,054,039 to Shieh, which are hereby incorporated in theirentireties.

[0006] An important limitation of electrochemical methods of measuringthe concentration of a chemical in blood is the effect of confoundingvariables on the diffusion of analyte and the various active ingredientsof the reagent. For example, the geometry and state of the blood samplemust correspond closely to that upon which the signal-to-concentrationmapping function is based.

[0007] The geometry of the blood sample is typically controlled by asample-receiving portion of the testing apparatus. In the case of bloodglucose meters, for example, the blood sample is typically placed onto adisposable test strip that plugs into the meter. The test strip may havea sample chamber (capillary fill space) to define the geometry of thesample. Alternatively, the effects of sample geometry may be limited byassuring an effectively infinite sample size. For example, theelectrodes used for measuring the analyte may be spaced closely enoughso that a drop of blood on the test strip extends substantially beyondthe electrodes in all directions. Ensuring adequate coverage of themeasurement electrodes by the sample, however, is an important factor inachieving accurate test results. This has proven to be problematic inthe past, particularly with the use of capillary fill spaces.

[0008] Other examples of limitations to the accuracy of blood glucosemeasurements include variations in blood composition or state (otherthan the aspect being measured). For example, variations in hematocrit(concentration of red blood cells), or in the concentration of otherchemicals in the blood, can effect the signal generation of a bloodsample. Variations in the temperature of blood samples is yet anotherexample of a confounding variable in measuring blood chemistry.

[0009] Thus, a system and method are needed that accurately measureblood glucose, even in the presence of confounding variables, includingvariations in temperature, hematocrit, and the concentrations of otherchemicals in the blood. A system and method are also needed to ensureadequate coverage of the measurement electrodes by the sample,particularly in capillary fill devices. A system and method are likewiseneeded that accurately measure an analyte in a fluid. It is an object ofthe present invention to provide such a system and method.

SUMMARY OF THE INVENTION

[0010] In one embodiment of the present invention, a method fordetermining a glucose concentration of a blood sample is disclosed,comprising the steps of a) applying a signal having an AC component tothe blood sample; b) measuring an AC phase angle response to the signal;and c) determining the glucose concentration using the AC phase angleresponse.

[0011] In another embodiment of the present invention, a method ofdetermining a glucose concentration of a biological fluid sample isdisclosed, comprising (a) applying a signal having an AC component tothe sample; (b) measuring an AC phase angle response to the signal; anddetermining the glucose concentration based upon the AC phase angleresponse and a predetermined correlation between the AC phase angleresponse and the glucose concentration.

[0012] In yet another embodiment of the present invention, a method ofdetermining a glucose concentration of a test sample is disclosedcomprising (a) applying a signal having an AC component to the sample;(b) measuring an AC phase angle response to the signal; and (c)determining the glucose concentration using the first AC phase angleresponse and a predetermined compensation factor.

[0013] In another embodiment of the present invention, a method fordetermining a hematocrit value of a blood sample is disclosed,comprising the steps of a) applying at least one signal having an ACcomponent to the blood sample; b) measuring at least one AC phase angleresponse to respective ones of the at least one signal; and c)determining the hematocrit value using the at least one AC phase angleresponse.

[0014] In another embodiment of the present invention, a method fordetermining a hematocrit value of a blood sample is disclosed,comprising the steps of (a) applying a first signal having an ACcomponent to the blood sample, the first signal having a firstfrequency; (b) measuring a first AC phase angle response to the firstsignal; (c) applying a second signal having an AC component to the bloodsample, the second signal having a second frequency; (d) measuring asecond AC phase angle response to the second signal; and (e) determiningthe hematocrit value based at least in part upon the first phase angleresponse and the second phase angle response.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The invention will be further described, by way of example only,with reference to the accompanying drawings, in which:

[0016]FIG. 1 is a diagram of a first embodiment excitation signalsuitable for use in a system and method according to the presentinvention, having a serially-applied AC component and DC component.

[0017]FIG. 2 is a diagram of a second embodiment excitation signalsuitable for use in a system and method according to the presentinvention, having a simultaneously-applied AC component and DCcomponent.

[0018] FIGS. 3A-B illustrate a first embodiment test strip of thepresent invention.

[0019]FIG. 4 is a diagram of an excitation signal utilized in the testof Example 1.

[0020]FIG. 5 is a plot of the correlation coefficient r² (glucose vs. DCcurrent) versus Read Time for the test of Example 1 with no incubationtime.

[0021]FIG. 6 is a plot of the correlation coefficient r² (glucose vs. DCcurrent) versus Read Time for the test of Example 1 with varyingincubation time.

[0022]FIG. 7 is a plot of AC admittance versus hematocrit for the testof Example 2.

[0023]FIG. 8 is a plot of uncompensated DC current versus glucose forthe test of Example 2.

[0024]FIG. 9 is a plot of the predicted glucose response versus theactual glucose response for the test of Example 2.

[0025]FIG. 10 is a diagram of an excitation signal utilized in the testof Example 3.

[0026]FIG. 11 is a plot of the AC phase angle versus reference glucosefor the test of Example 3.

[0027]FIG. 12 is a plot of the predicted glucose response versus theactual glucose response for the test of Example 3.

[0028]FIG. 13 is a diagram of an excitation signal utilized in the testof Example 4.

[0029]FIG. 14 is a plot of AC admittance versus hematocrit(parametrically displayed with temperature) for the test of Example 4.

[0030]FIG. 15 is a plot of the uncompensated DC response versus actualglucose for the test of Example 4.

[0031]FIG. 16 is a plot of the predicted glucose response versus actualglucose response for the test of Example 4.

[0032] FIGS. 17A-B illustrate a second embodiment test strip of thepresent invention.

[0033]FIG. 18 is a plot parametrically illustrating the correlationcoefficient r² between the DC current response and glucose level as ReadTime varies for three combinations of temperature and hematocrit in thetest of Example 5.

[0034]FIG. 19 is a diagram of the excitation signal utilized in the testof Example 5.

[0035]FIG. 20 is a plot of AC admittance versus hematocrit astemperature is parametrically varied in the test of Example 5.

[0036]FIG. 21 is a plot of AC admittance phase angle versus hematocritas temperature is parametrically varied in the test of Example 5.

[0037]FIG. 22 is a plot of the uncompensated DC response versus actualglucose for the test of Example 5.

[0038]FIG. 23 is a plot of the predicted glucose response versus actualglucose response for the test of Example 5.

[0039]FIG. 24 is a diagram of the excitation signal utilized in the testof Example 6.

[0040]FIG. 25 is a plot of the correlation coefficient r² betweenhematocrit and DC response current plotted against hematocrit in thetest of Example 6.

[0041]FIG. 26 is a plot of AC admittance phase angle versus hematocritfor the test of Example 6.

[0042]FIG. 27 is a plot of the uncompensated DC response versus actualglucose for the test of Example 6.

[0043]FIG. 28 is a plot of the compensated DC response versus actualglucose for a 1.1 second Total Test Time of Example 6.

[0044]FIG. 29 is a plot of the compensated DC response versus actualglucose for a 1.5 second Total Test Time of Example 6.

[0045]FIG. 30 is a plot of the compensated DC response versus actualglucose for a 1.9 second Total Test Time of Example 6.

[0046]FIG. 31 is a table detailing the heights and widths of thecapillary fill channels used in the test devices of Example 8, as wellas schematic diagrams of convex and concave sample flow fronts in acapillary fill space.

[0047] FIGS. 32A-C are schematic plan views of a test strip illustratingthe potential for biased measurement results when a concave flow frontencounters a prior art dose sufficiency electrode.

[0048]FIG. 33 is a schematic plan view of a test strip of the presentinvention having a pair of perpendicular dose sufficiency electrodesthat are independent from the measurement electrodes.

[0049] FIGS. 34A-B are schematic plan views of the test strip of FIG. 33containing samples with convex and concave flow fronts, respectively.

[0050] FIGS. 35A-B are schematic plan views of a test strip of thepresent invention having a pair of parallel dose sufficiency electrodesthat are independent from the measurement electrodes.

[0051]FIG. 36 is a schematic plan view of the test strip of FIG. 35,schematically illustrating the electric field lines that communicatebetween the electrode gap when the electrodes are covered with sample.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0052] For the purposes of promoting an understanding of the principlesof the invention, reference will now be made to the embodimentillustrated in the drawings, and specific language will be used todescribe that embodiment. It will nevertheless be understood that nolimitation of the scope of the invention is intended. Alterations andmodifications in the illustrated device, and further applications of theprinciples of the invention as illustrated therein, as would normallyoccur to one skilled in the art to which the invention relates arecontemplated, are desired to be protected. In particular, although theinvention is discussed in terms of a blood glucose meter, it iscontemplated that the invention can be used with devices for measuringother analytes and other sample types. Such alternative embodimentsrequire certain adaptations to the embodiments discussed herein thatwould be obvious to those skilled in the art.

[0053] The entire disclosure of U.S. provisional applications titledDEVICES AND METHODS RELATING TO ELECTROCHEMICAL BIOSENSORS (Serial No.60/480,243, filed Jun. 20, 2003) and DEVICES AND METHODS RELATING TOANALYTE SENSOR (Serial No. 60/480,397, Filed Jun. 20, 2003) are herebyincorporated by reference in their entireties.

[0054] A system and method according to the present invention permit theaccurate measurement of an analyte in a fluid. In particular, themeasurement of the analyte remains accurate despite the presence ofinterferants, which would otherwise cause error. For example, a bloodglucose meter according to the present invention measures theconcentration of blood glucose without error that is typically caused byvariations in the temperature and the hematocrit level of the sample.The accurate measurement of blood glucose is invaluable to theprevention of blindness, loss of circulation, and other complications ofinadequate regulation of blood glucose in diabetics. An additionaladvantage of a system and method according to the present invention isthat measurements can be made much more rapidly and with much smallersample volumes, making it more convenient for the diabetic person tomeasure their blood glucose. Likewise, accurate and rapid measurement ofother analytes in blood, urine, or other biological fluids provides forimproved diagnosis and treatment of a wide range of medical conditions.

[0055] It will be appreciated that electrochemical blood glucose meterstypically (but not always) measure the electrochemical response of ablood sample in the presence of a reagent. The reagent reacts with theglucose to produce charge carriers that are not otherwise present inblood. Consequently, the electrochemical response of the blood in thepresence of a given signal is intended to be primarily dependent uponthe concentration of blood glucose. Secondarily, however, theelectrochemical response of the blood to a given signal is dependentupon other factors, including hematocrit and temperature. See, forexample, U.S. Pat. Nos. 5,243,516; 5,288,636; 5,352,351; 5,385,846; and5,508,171, which discuss the confounding effects of hematocrit on themeasurement of blood glucose, and which are hereby incorporated byreference in their entireties. In addition, certain other chemicals caninfluence the transfer of charge carriers through a blood sample,including, for example, uric acid, bilirubin, and oxygen, therebycausing error in the measurement of glucose.

[0056] A preferred embodiment system and method for measuring bloodglucose according to the present invention operates generally by usingthe signal-dependence of the contribution of various factors to theimpedance (from which admittance and phase angle may be derived) of ablood sample. Because the contribution of various factors to theimpedance of a blood sample is a function of the applied signal, theeffects of confounding factors (that is, those other than the factorssought to be measured) can be substantially reduced by measuring theimpedance of the blood sample to multiple signals. In particular, theeffects of confounding factors, (primarily temperature and hematocrit,but also including chemical interferants such as oxygen), contributeprimarily to the resistivity of the sample, while the glucose-dependentreaction contributes primarily to the capacitance. Thus, the effects ofthe confounding factors can be eliminated by measuring the impedance ofthe blood sample to an AC excitation, either alone or in combinationwith a DC excitation. The impedance (or the impedance derived admittanceand phase information) of the AC signal is then used to correct the DCsignal or AC derived capacitance for the effects of interferants.

[0057] It will be appreciated that measurements at sufficiently high ACfrequencies are relatively insensitive to the capacitive component ofthe sample's impedance, while low frequency (including DC) measurementsare increasingly (with decreasing frequency) sensitive to both theresistive and the capacitive components of the sample's impedance. Theresistive and capacitive components of the impedance can be betterisolated by measuring the impedance at a larger number of frequencies.However, the cost and complexity of the meter increases as the number ofmeasurements increases and the number of frequencies that need to begenerated increases. Thus, in the presently preferred embodiment, theimpedance may be measured at greater than ten frequencies, butpreferably at between two and ten frequencies, and most preferably atbetween two and five frequencies.

[0058] As used herein, the phrase “a signal having an AC component”refers to a signal which has some alternating potential (voltage)portions. For example, the signal may be an “AC signal” having 100%alternating potential (voltage) and no DC portions; the signal may haveAC and DC portions separated in time; or the signal may be AC with a DCoffset (AC and DC signals superimposed).

[0059] Sample Measurement with Successive AC and DC Signals

[0060]FIG. 1 illustrates a preferred embodiment excitation signalsuitable for use in a system and method according to the presentinvention, indicated generally at 100, in which DC excitation and fourfrequencies of AC excitation are used. FIG. 1 also illustrates a typicalresponse to the excitation when the excitation is applied to a sample ofwhole blood mixed with an appropriate reagent, the response indicatedgenerally at 102. A relatively high frequency signal is applied,starting at time 101. In the preferred embodiment the frequency isbetween about 10 kHz and about 20 kHz, and has an amplitude betweenabout 12.4 mV and about 56.6 mV. A frequency of 20 kHz is used in theexample of FIG. 1. Those skilled in the art will appreciate that thesevalues may be optimised to various parameters such as cell geometry andthe particular cell chemistry.

[0061] At time 110 a test strip is inserted into the meter and severalpossible responses to the insertion of the test strip into the glucosemeter are shown. It will be appreciated that the test strip may also beinserted before the excitation signal 100 is initiated (i.e. before time101); however, the test strip itself may advantageously be tested as acontrol for the suitability of the strip. It is therefore desirable thatthe excitation signal 100 be initiated prior to test strip insertion.For example, relatively large current leakage, as shown at 112, mayoccur if the strip is wet, either because the test strip was pre-dosed,or due to environmental moisture. If the test strip has been pre-dosedand permitted to largely or completely dry out, an intermediate currentleakage may occur, as shown at 114. Ideally, insertion of the test stripwill cause no or negligible leakage current due to an expected absenceof charge carriers between the test electrodes, as shown at 116.Measured current leakage above a predetermined threshold level willpreferably cause an error message to be displayed and prevent the testfrom continuing.

[0062] Once a suitable test strip has been inserted, the user doses thestrip, as shown at time 120. While the blood sample is covering theelectrodes the current response will rapidly increase, as the glucosereacts with the reagent and the contact area increases to maximum. Theresponse current will reach a stable state, which indicates theimpedance of the sample at this frequency. Once this measurement is madeand recorded by the test meter, the excitation frequency is then steppeddown to about 10 kHz in the preferred embodiment, as shown at time 130.Another measurement is made and recorded by the test meter, and thefrequency is stepped down to about 2 kHz in the preferred embodiment, asshown at 140. A third measurement is made and recorded by the test meterat this frequency. A fourth measurement is made at about 1 kHz in thepreferred embodiment, as shown at 150. In the preferred embodiment,measurements are taken at regular intervals (e.g. 10 points per cycle).It will be appreciated that the stable state response may be measured ascurrent or voltage (preferably both magnitude and phase) and theimpedance and/or admittance can be calculated therefrom. Although thepresent specification and claims may refer alternately to the ACresponse as impedance or admittance (magnitude and/or phase),resistance, conductivity, current or charge, and to the DC response ascurrent, charge, resistance or conductivity, those skilled in the artwill recognize that these measures are interchangeable, it only beingnecessary to adjust the measurement and correction mathematics toaccount for which measure is being employed. In the preferredembodiment, the test meter applies a voltage to one electrode andmeasures the current response at the other electrode to obtain both theAC and DC response.

[0063] In certain alternative embodiments measurements are made at feweror more frequencies. Preferably measurements are made at at least two ACfrequencies at least an order of magnitude apart. If more than two ACfrequencies are used, then it is preferable that the highest and lowestfrequencies be at least an order of magnitude apart.

[0064] It will be appreciated that various waveforms may be used in anAC signal, including, for example, sinusoidal, trapezoidal, triangle,square and filtered square. In the presently preferred embodiment the ACsignal has a filtered square waveform that approximates a sine wave.This waveform can be generated more economically than a true sine wave,using a square wave generator and one or more filters.

[0065] Once all four AC measurements are made, the signal is preferablybriefly reduced to zero amplitude, as shown at 160. The DC excitation isthen begun, as shown at 170. The amplitude of the DC excitation isadvantageously selected based on the reagent being used, in order tomaximise the resulting response or response robustness. For example, ifferricyanide is being used in a biamperometry system, the DC amplitudeis preferably about 300 mV. For another example, if a nitrosoanilinederivative is being used in a biamperometry system, the DC amplitude ispreferably about 500-550 mV. In the alternative, if a third referenceelectrode is used, the DC applitude is preferably 600 mV (versus thesilver/silver chloride reference electrode) for ferricyanide, and 40-100mV (versus the silver/silver chloride reference electrode) fornitrosoaniline derivative. During DC excitation, measurements arepreferably made at a rate of 100 pts/sec. The current response willfollow a decay curve (known as a Cottrell curve), as the reaction islimited by the diffusion of unreacted glucose next to the workingelectrode. The resulting stable-state amplitude (measured or projected)is used to determine a glucose estimation of the sample, as is known inthe art. A corrected estimation is then determined that corresponds moreclosely to the concentration of glucose in the blood, by using theimpedance of the sample to the AC signal to correct for the effects ofinterferants, as explained in greater detail hereinbelow.

[0066] It will be appreciated that a method according to the presentinvention may also be used to measure the concentration of otheranalytes and in other fluids. For example, a method according to thepresent invention may be used to measure the concentration of amedically significant analyte in urine, saliva, spinal fluid, etc.Likewise, by appropriate selection of reagent a method according to thepresent invention may be adapted to measure the concentration of, forexample, lactic acid, hydroxybutyric acid, etc.

[0067] Sample Measurement with Simultaneously Applied AC and DC Signals

[0068] It will be appreciated that at least some of the applied DC andAC components can also be applied simultaneously. FIG. 2 illustrates anexcitation signal suitable for use in a system and method according tothe present invention in which some of the AC and DC components areapplied simultaneously, indicated generally at 200, and havingcorresponding events numbered correspondingly to FIG. 1 (so, forexample, the signal 200 is initiated at time 201, and a strip isinserted at time 210, etc.). As with the signal 100, the signal 200 hasa frequency of about 10-20 kHz and an amplitude of about 12.4-56.6 mV.However, after the strip has been dosed, as shown at time 220, a DCoffset is superimposed, as shown at 270. Typical AC and DC responses areshown in FIG. 2. The AC and DC responses are measured simultaneously andmathematically deconvoluted and used to determine the impedance(admittance magnitude and phase) and the amperometric or coulometricresponse.

[0069] A system for measuring blood glucose according to the presentinvention advantageously employs a blood glucose meter and test stripsgenerally similar to those used in prior art systems, such as thosecommercially available from Roche Diagnostics, and such as are describedin U.S. Pat. Nos. 6,270,637; and 5,989,917, which are herebyincorporated in their entireties. These test strips provide apparatihaving a sample cell in which the blood sample is received for testing,and electrodes disposed within the sample cell through which theexcitation signal is provided and the measurements are made. Thoseskilled in the art will appreciate that these test strips and meters mayadvantageously be used for the measurement of glucose in blood, but thatother apparati may be more suitable for the measurement of otheranalytes or other biological fluids when practising the presentinvention.

[0070] A suitable glucose meter may be adapted from such known meters bythe addition of electronic circuitry that generates and measures signalshaving AC and DC components, such as those described hereinabove, and bybeing programmed to correct the DC measurement using the ACmeasurement(s), as described in greater detail hereinbelow. It will beappreciated that the specific geometry and chemistry of the test stripscan cause variations in the relationships between the concentration ofglucose, hematocrit, and temperature, and the impedance of a sample.Thus, a given combination of test strip geometry and chemistry must becalibrated, and the meter programmed with the corresponding algorithm.The present invention comprehends the application of excitation signalsin any order and combination. For example, the present inventioncomprehends the application of 1) AC only, 2) AC then DC, 3) AC then DCthen AC, 4) DC then AC, and 5) AC with a DC offset, just to name a fewof the possible permutations.

[0071] The use of the complex AC impedance measurement data to correctfor the effects of interferants on the DC measurement is advantageouslyillustrated by the following series of examples. These examplesillustrate how the principles of the present invention can facilitateimprovements in accuracy and test speed when measuring the concentrationof an analyte in a test specimen. Although the following examples dealwith correcting for the interfering effects of hematocrit andtemperature on blood glucose determinations, those skilled in the artwill recognize that the teachings of the present invention are equallyuseful for correcting for the effects of other interferants in bothblood glucose measurements and in the measurement of other analytes.Furthermore, the present specification and claims refer to steps such as“determine the hematocrit value” and “determine the temperature,” etc.To use the hematocrit value as an example, it is intended that suchstatements include not only determining the actual hematocrit value, butalso a hematocrit correction factor vs. some nominal point. In otherwords, the process may never actually arrive at a number equal to thehematocrit value of the sample, but instead determine that the sample'shematocrit differs from a nominal value by a certain amount. Bothconcepts are intended to be covered by statements such as “determine thehematocrit value.”

EXAMPLE 1 DC-Only Measurement Dose Response Study

[0072] The measurements made in Example 1 were achieved using the teststrip illustrated in FIGS. 3A-B and indicated generally at 300. The teststrip 300 includes a capillary fill space containing a relatively thickfilm reagent and working and counter electrodes, as described in U.S.Pat. No. 5,997,817, which is hereby incorporated by reference. The teststrip 300 is commercially available from Roche Diagnostics Corporation(Indianapolis, Ind.) under the brand name Comfort Curve®. Theferricyanide reagent used had the composition described in Tables I andII. TABLE I Reagent Mass Composition - Prior to Dispense and Drying Massfor Component % w/w 1 kg solid Polyethylene oxide (300 kDa) 0.8400%8.4000 g solid Natrosol 250M 0.0450% 0.4500 g solid Avicel RC-591F0.5600% 5.6000 g solid Monobasic potassium 1.2078% 12.0776 g  phosphate(annhydrous) solid Dibasic potassium 2.1333% 21.3327 g  phosphate(annhydrous) solid Sodium Succinate hexahydrate 0.6210% 6.2097 g solidQuinoprotein glucose 0.1756% 1.7562 g dehydrogenase (EnzC#: 1.1.99.17)solid PQQ 0.0013% 0.0125 g solid Trehalose 2.0000% 20.0000 g  solidPotassium Ferricyanide 5.9080% 59.0800 g  solid Triton X-100 0.0350%0.3500 g solvent Water 86.4731%  864.7313 g 

[0073] TABLE II Reagent Layer Composition - After Drying Mass perComponent % w/w Sensor solid Polyethylene oxide (300 kDa) 6.2099%38.6400 ug  solid Natrosol 250M 0.3327% 2.0700 ug solid Avicel RC-591F4.1399% 25.7600 ug  solid Monobasic potassium phosphate 8.9286% 55.5568ug  (annhydrous) solid Dibasic potassium phosphate 15.7706%  98.1304 ug (annhydrous) solid Sodium Succinate hexahydrate 4.5906% 28.5646 ug solid Quinoprotein glucose 1.2983% 8.0784 ug dehydrogenase (EnzC#:1.1.99.17) solid PQQ 0.0093% 0.0576 ug solid Trehalose 14.7854%  92.0000ug  solid Potassium Ferricyanide 43.6760%  271.7680 ug  solid TritonX-100 0.2587% 1.6100 ug

[0074] In the measurements, blood samples were applied to test strip 300and the excitation potentials illustrated in FIG. 4 were applied to theelectrodes. The excitation comprised a 2 kHz 40 mV_(rms) (56.56 mV peak)AC signal applied between 0 seconds and approximately 4.5 seconds aftersample application, followed by a 300 mV DC signal applied thereafter.For the calculations of this example, however, only the DC measurementdata was analyzed.

[0075] In order to determine the minimum needed DC excitation time, a“dose response” study was performed, in which glycollyzed (glucosedepleted) blood was divided into discrete aliquots and controlled levelsof glucose were added to obtain five different known levels of glucosein the blood samples. The resulting DC current profile was then examinedas two parameters were varied. The first parameter was the IncubationTime, or the time between the detection of the blood sample beingapplied to the test strip 300 and the application of the DC potential tothe test strip 300. The second parameter to be varied was the Read Time,or the time period after application of the DC potential and themeasurement of the resulting current. The length of time betweendetection of the blood sample being applied to the test strip to thetaking of the last measurement used in the concentration determinationcalculations is the Total Test Time. In this study, therefore, the sumof the Incubation Time and the Read Time is the Total Test Time. Theresults of this study are illustrated in FIGS. 5 and 6.

[0076] In FIG. 5, the DC response was measured with no incubation time(Read Time=Total Test Time). FIG. 5 plots the correlation coefficient r²versus Read Time. As can be seen, the correlation exceeds 0.95 within1.0 second. In FIG. 6, the DC response was measured with varyingIncubation Time. When an Incubation Time is provided (even an IncubationTime as short as two (2) seconds), the r² value rose to over 0.99 in 0.5seconds or less after application of the DC potential.

[0077] The barrier to implementation of such fast test times in aconsumer glucose test device, however, is the variation from bloodsample to blood sample of the level of interference from the presence ofblood cells in the sample. The hematocrit (the percentage of the volumeof a blood sample which is comprised of cells versus plasma) varies fromindividual to individual. The interference effect of hematocrit on suchmeasurements is fairly complex. In the tests of Example 1, however, allsamples contained the same level of hematocrit. With no variablehematocrit influence at the different glucose levels, the hematocritterm cancels out in the correlation figures.

EXAMPLE 2 Combined AC and DC Measurement of Capillary Blood Samples

[0078] The measurements made in Example 2 were also achieved using thetest strip illustrated in FIGS. 3A-B and indicated generally at 300. Asdescribed above, the test strip 300 includes a capillary fill spacecontaining a relatively thick film reagent and working and counterelectrodes, as described in U.S. Pat. No. 5,997,817, which is herebyincorporated herein by reference.

[0079] In the measurements, capillary blood samples from variousfingerstick donors were applied to test strip 300 and the excitationpotentials illustrated in FIG. 4 were applied to the electrodes. Theexcitation comprised a 2 kHz 40 mV_(rms) AC signal applied between 0seconds and approximately 4.5 seconds after sample application, followedby a 300 mV DC signal applied thereafter.

[0080] In this Example 2, the AC response of the sample was derived asadmittance (the inverse of impedance). The admittance response isproportionate to the hematocrit level of the sample in a temperaturedependent manner. The relationship between admittance, hematocrit andtesting temperature is illustrated in FIG. 7. The data used for theadmittance charted in FIG. 7 is the last admittance measurement made foreach sample during the AC portion of the excitation illustrated in FIG.4.

[0081] Regression analysis of this data allows admittance, hematocritand temperature to be related according to the following formula:

H _(est) =c ₀ +c ₁ Y _(2 kHz) +c ₂ dT  (Equation 1)

[0082] Using this relationship to predict the blood hematocrit isaccomplished using test temperature data reported by the temperaturesensor in the meter and the measured admittance. In Equation 1, c₀, c₁and c₂ are constants, dT is the deviation in temperature from a centerdefined as “nominal” (24° C. for example), and H_(est) is the estimateddeviation in hematocrit from a similar “nominal” value. For the presentpurposes, the actual hematocrit value is not necessary, and it isgenerally preferred to produce a response which is proportionate butcenters around a nominal hematocrit. Thus, for a 70% hematocrit, thedeviation from a nominal value of 42% would be 28%, while conversely fora 20% hematocrit the deviation from that same nominal value would be−22%.

[0083] By using the AC admittance measurement to estimate the hematocritlevel using Equation 1, the accuracy of the DC glucose response can begreatly improved by combining the estimated hematocrit, temperature andDC response to correct for the hematocrit interference in the DCresponse as follows:

PRED=(a ₀ +hct ₁ H _(est) +hct ₂ H _(est) ² +tau ₁ dT+tau ₂ dT ²)+(a ₁DC)(1+hct ₃ H _(est) +hct ₄ H _(est) ²)(1+tau ₃ dT+tau ₄ dT²)  (Equation 2)

[0084] where DC is the measured glucose current response to the appliedDC signal and PRED is the compensated (predicted) glucose responsecorrected for the effects of hematocrit and temperature. The constants(a₀, hct₁, hct₂, tau₁, tau₂, a₁, hct₃, hct₄, tau₃ and tau₄) in Equation2 can be determined using regression analysis, as is known in the art.

[0085]FIG. 8 illustrates the uncompensated 5.5 second DC glucoseresponse of all of the capillary blood samples as temperature varies(ignoring the AC measurement data). As will be appreciated, there is awide variation in the DC current response as temperature and hematocritvary. FIG. 9 illustrates the correlation between the actual bloodglucose level of the sample versus the predicted response using Equation2. As can be seen, when the DC response is compensated for hematocritlevels using the AC response data, r² values of 0.9404 to 0.9605 areachieved with a Total Test Time of 5.5 seconds.

EXAMPLE 3 Use of AC Phase Angle to Estimate Blood Glucose Levels andHematocrit

[0086] The measurements made in Example 3 were also achieved using thetest strip illustrated in FIGS. 3A-B and indicated generally at 300. Asdescribed above, the test strip 300 includes a capillary fill spacecontaining a relatively thick film reagent and working and counterelectrodes, as described in U.S. Pat. No. 5,997,817, which is herebyincorporated by reference. Because hematocrit levels from capillaryblood samples typically vary only between 30%-50%, spiked venous bloodsamples having a hematocrit range from 20%-70% were used for thisExample 3. Five levels of glucose, temperature (14, 21, 27, 36 and 42°C.) and hematocrit (20, 30, 45, 60 and 70%) were independently varied,producing a covariance study with 125 samples.

[0087] In the measurements, blood samples were applied to test strip 300and the excitation potentials illustrated in FIG. 10 were applied to theelectrodes. The excitation comprised a 2 kHz AC signal for approximately4.1 seconds, a 1 kHz AC signal for approximately 0.1 seconds, and a 200Hz signal for approximately 0.1 seconds. All three AC signals had anamplitude of 56.56 mV peak. No DC excitation was used in this example.The Total Test Time was 4.3 seconds from sample application time.

[0088] It was found that another component of the AC response, the phaseangle (particularly at lower frequencies, such as 200 Hz in this Example3), is also a function of the sample glucose level in the case of thistest strip and reagent. This relationship is demonstrated in FIG. 11,where the AC phase angle for each of the three test frequencies isplotted versus the reference glucose level. Regression analysis for eachof the three frequencies produces AC phase angle-to-reference glucoselevel r² correlation values of 0.9114 at 2 kHz, 0.9354 at 1 kHz, and0.9635 at 200 Hz. The present invention therefore comprehends the use ofthe AC phase angle to measure glucose levels. The AC excitationfrequency producing the measured phase angle is preferably 2 kHz orbelow, more preferably 1 kHz or below, and most preferably 200 Hz orbelow, but not including DC excitation.

[0089] The linearized relationship between the 200 Hz phase angleresponse and the blood glucose level is as follows:

P_(eff)=(Φ_(200 Hz)/⊖)^(−γ)  (Equation 3)

[0090] where P_(eff) is the effective phase, which is proportional toglucose, the terms ⊖ and γ are constants, and Φ is the measured AC phaseangle.

[0091] Using the same approach to compensate for temperature andhematocrit as used in Example 1 above (see Equations 1 and 2) produced apredictive algorithm as follows:

PRED=(a ₀ +hct ₁ H _(est) +hct ₂ H _(est) +tau ₁ dT+tau ₂ dT ²)+(a ₁ P_(eff))(1+hct ₃ H _(est) +hct ₄ H _(est) ²)(1+tau ₃ dT+tau ₄ dT²)  (Equation 4)

[0092] The resulting compensated (predicted) response PRED versusglucose for the 125 blood samples (each tested with eight test strips)is shown in FIG. 12. The r² correlation of the PRED response vs. knownglucose level, where all temperatures and all hematocrits are combined,is 0.9870. This Example 3 demonstrates again the value of ACmeasurements for compensating for interferants that reduce the accuracyof blood glucose measurements. Using an existing commercially availablesensor, the present invention yields a 4.3 second Total Test Time withan overall r² of 0.9870.

[0093] It was also determined that AC phase angle measurements canproduce hematocrit level measurements that are almost immune to theeffects of temperature variation. In another covariant study of 125samples (five glucose concentrations, five hematocrit concentrations andfive temperatures), each of the samples was tested using an excitationprofile of 20 kHz, 10 kHz, 2 kHz, 1 kHz and DC. The AC phase angle atvarious frequencies was related to glucose, hematocrit and temperatureusing linear regression to determine the coefficients of the followingformula at each of the four AC frequencies:

Phase=c ₀ +c ₁ Glu+c ₂ HCT+c ₃ Temp  (Equation 5)

[0094] where Glu is the known glucose concentration, HCT is the knownhematocrit concentration and Temp is the known temperature.

[0095] The determined coefficients revealed that the temperaturecoefficient (c₃) was essentially zero at 20 kHz and 10 kHz, cancellingtemperature from the equation at these frequencies. Furthermore, theglucose coefficient (c₁) is essentially zero at all of the ACfrequencies because, as explained hereinabove, the higher frequency ACimpedance measurements are largely unaffected by glucose levels and aretherefore useful for measuring the levels of interfering substances. Itwas therefore found that the hematocrit level could be determinedindependent of temperature and glucose level using only the AC phaseangle measurements. In a preferred embodiment, the hematocrit may bemeasured using the phase angle data from all four measured frequencies:

H _(est) =c ₀ +c ₁Φ_(20 kHz) +c ₂Φ_(10 kHz) +c ₃Φ_(2 kHz) +c₄Φ_(1 kHz)  (Equation 6)

[0096] Those skilled in the art will recognise that that thecoefficients can be empirically determined for any particular test striparchitecture and reagent chemistry. The present invention therefore maybe used to estimate hematocrit using only AC phase angle measurementspreferably made at at least one AC frequency, more preferably made at atleast two AC frequencies, and most preferably made at at least four ACfrequencies.

EXAMPLE 4 Combined AC and DC Measurement Using Nitrosoaniline Reagent

[0097] The measurements made in Example 4 were also achieved using thetest strip illustrated in FIGS. 3A-B and indicated generally at 300. Asdescribed above, the test strip 300 includes a capillary fill spacecontaining a relatively thick film reagent and working and counterelectrodes, as described in U.S. Pat. No. 5,997,817, which is herebyincorporated by reference. The test strip was modified from thatdescribed in U.S. Pat. No. 5,997,817, however, by the use of a differentreagent. The nitrosoaniline reagent used had the composition describedin Tables III and IV. TABLE III Reagent Mass Composition - Prior toDispense and Drying Mass for Component % w/w 1 kg solid Polyethyleneoxide (300 kDa) 0.8054% 8.0539 g solid Natrosol 250M 0.0470% 0.4698 gsolid Avicel RC-591F 0.5410% 5.4104 g solid Monobasic potassium 1.1437%11.4371 g  phosphate (annhydrous) solid Dibasic potassium 1.5437%15.4367 g  phosphate (annhydrous) solid Disodium Succinate hexahydrate0.5876% 5.8761 g solid Potassium Hydroxide 0.3358% 3.3579 g solidQuinoprotein glucose 0.1646% 1.6464 g dehydrogenase (EnzC#: 1.1.99.17)solid PQQ 0.0042% 0.0423 g solid Trehalose 1.8875% 18.8746 g  solidMediator 31.1144 0.6636% 6.6363 g solid Triton X-100 0.0327% 0.3274 gsolvent Water 92.2389%  922.3888 g 

[0098] TABLE IV Reagent Layer Composition - After Drying Mass perComponent % w/w Sensor solid Polyethylene oxide (300 kDa) 10.3829% 37.0480 ug solid Natrosol 250M 0.6057%  2.1611 ug solid Avicel RC-591F6.9749% 24.8877 ug solid Monobasic potassium 14.7445%  52.6107 ugphosphate (annhydrous) solid Dibasic potassium 19.9006%  71.0087 ugphosphate (annhydrous) solid Disodium Succinate hexahydrate 7.5753%27.0299 ug solid Potassium Hydroxide 4.3289% 15.4462 ug solidQuinoprotein glucose dehydrogenase 2.1225%  7.5734 ug (EnzC#: 1.1.99.17)solid PQQ 0.0546%  0.1947 ug solid Trehalose 24.3328%  86.8243 ug solidMediator BM 31.1144 8.5553% 30.5268 ug solid Triton X-100 0.4220% 1.5059 ug

[0099] The method for the manufacture of the glucose biosensor for thisExample 4 is the same in all respects as disclosed in U.S. Pat. No.5,997,817 except for the manufacture of the reagent. A protocol for thepreparation of the preferred embodiment nitrosoaniline reagent is asfollows:

[0100] Step 1: Prepare a buffer solution by adding 1.54 g of dibasicpotassium phosphate (anhydrous) to 43.5 g of deionized water. Mix untilthe potassium phosphate is dissolved.

[0101] Step 2: To the solution from step 1, add 1.14 g of monobasicpotassium phosphate and mix until dissolved.

[0102] Step 3: To the solution from step 2, add 0.59 g of disodiumsuccinate (hexahydrate) and mix until dissolved.

[0103] Step 4: Verify that the pH of the solution from step 3 is6.7+/−0.1. Adjustment should not be necessary.

[0104] Step 5: Prepare a 5 g aliquot of the solution from step 4, and tothis add 113 kilounits (by DCIP assay) of the apoenzyme of quinoproteinglucose dehydrogenase (EC#: 1.1.99.17). This is approximately 0.1646 g.Mix, slowly, until the protein is dissolved.

[0105] Step 6: To the solution from step 5, add 4.2 milligrams of PQQand mix for no less than 2 hours to allow the PQQ and the apoenzyme toreassociate in order to provide functional enzyme.

[0106] Step 7: To the solution from step 4, add 0.66 g of the mediatorprecursor, N,N-bis(hydroxyethyl)-3-methoxy-4-nitrosoaniline(hydrochloride) (BM 31.1144). Mix until dissolved (this solution willhave a greenish black coloration).

[0107] Step 8: Measure the pH of the solution from step 7 and adjust thepH to a target of 7.0+/−0.1. Normally this is accomplished with 1.197 gof 5N potassium hydroxide. Because the specific amount of potassiumhydroxide may vary as needed to reach the desired pH, generallydeviations in mass from the 1.197 g are made up from an aliquot of 3.309g deionized water which is also added at this step.

[0108] Step 9: Prepare a solution of Natrosol 250M (available fromAqualon), by slowly sprinkling 0.047 g over 44.57 g of deionized waterwhich is mixed (using a rotary mixer and blade impeller) at a rate ofapproximately 600 rpm in a vessel of sufficient depth such that therotor blades are not exposed nor the solution running over. Mix untilthe Natrosol is completely dissolved.

[0109] Step 10: Prepare a suspension of Avicel RC-591F (available fromFMS), by slowly sprinkling 0.54 g onto the surface of the solution fromstep 9, mixing at a rate of approximately 600 rpm for not less than 60minutes before proceeding.

[0110] Step 11: To the suspension from step 10, gradually add 0.81 g ofPolyethylene oxide of 300 kDa mean molecular weight while mixing andcontinue to mix for not less than 60 minutes before proceeding.

[0111] Step 12: Gradually add the solution from step 8 to the suspensionfrom step 11 while mixing. Reduce the mixing rate to 400 rpm.

[0112] Step 13: To the reagent from step 12, add 1.89 g of Trehalose andcontinue mixing for not less than 15 minutes.

[0113] Step 14: To the reagent from step 13, add 32.7 mg of Triton X-100(available from Roche Diagnostics) and continue mixing.

[0114] Step 15: To the reagent from step 14, add the enzyme solutionfrom step 6. Mix for no less than 30 minutes. At this point the reagentis complete. At room teperature the wet reagent mass is consideredacceptable for use for 24 hours.

[0115] Spiked venous blood samples were used. Five levels of glucose,four temperatures (19, 23, 32 and 38° C.) and five levels of hematocrit(20, 30, 45, 60 and 70%) were independently varied, producing acovariance study with 100 samples. 16 test strips 300 were tested foreach unique combination of glucose, temperature and hematocrit. Theblood samples were applied to test strip 300 and the excitationpotentials illustrated in FIG. 13 were applied to the electrodes. Theexcitation comprised a 3.2 kHz AC signal for approximately 4.0 seconds,a 2.13 kHz AC signal for approximately 0.1 seconds, a 1.07 kHz AC signalfor approximately 0.1 seconds, a 200 Hz AC signal for approximately 0.1seconds, a 25 Hz AC signal for approximately 0.1 seconds, followed by aDC signal of 550 mV for approximately 1.0 second. All four AC signalshad an amplitude of 56.56 mV peak. The Total Test Time was 5.5 secondsfrom sample application time.

[0116] In this Example 4, the AC response of the sample was derived asadmittance (the inverse of impedance). The admittance response isproportionate to the hematocrit level of the sample in a temperaturedependent manner. The relationship between admittance, hematocrit andtesting temperature is illustrated in FIG. 14. As compared to the teststrip architecture of Example 2, the orthogonality of the temperatureand hematocrit influence on glucose was not as strong in this Example 4,therefore a cross product term (T×HCT) was added to the admittanceregression formula used in FIG. 14. The data used for the admittancecharted in FIG. 14 is the last admittance measurement made for eachsample during the 3.2 kHz AC portion of the excitation illustrated inFIG. 13.

[0117] Regression analysis of this data allows admittance, hematocritand temperature to be related according to the following formula:

H _(est)=(Y _(3.2 kHz) +c ₀ +c ₁ dT)/(c ₂ dT+c ₃)  (Equation 7)

[0118] It was determined that the admittance measurement made at 3.2 kHzwas best correlated with hematocrit for this test system. Using thisrelationship to predict the blood hematocrit is accomplished using testtemperature data reported by the temperature sensor in the meter and themeasured admittance. In Equation 7, c₀, c₁, c₂ and C₃ are constants, dTis the deviation in temperature from a center defined as “nominal” (24°C. for example), and H_(est) is the estimated deviation in hematocritfrom a similar “nominal” value. For the present purposes, the actualhematocrit value is not necessary, and it is generally preferred toproduce a response which is proportionate but centers around a nominalhematocrit. Thus, for a 70% hematocrit, the deviation from a nominalvalue of 42% would be 28%, while conversely for a 20% hematocrit thedeviation from the same nominal value would be −22%.

[0119] By using the AC admittance measurement to estimate the hematocritlevel using Equation 7, the accuracy of the DC glucose response can begreatly improved by combining the estimated hematocrit, temperature andDC response to correct for the hematocrit interference in the DCresponse as follows (same as Equation 2 above):

PRED=(a ₀ +hct ₁ H _(est) +hct ₂ H _(est) ² +tau ₁ dT+tau ₂ dT ²)+(a ₁DC)(1+hct ₃ H _(est) +hct ₄ H _(est) ²)(1+tau ₃ dT+tau ₄ dT²)  (Equation 8)

[0120] The constants in Equation 8 can be determined using regressionanalysis, as is known in the art.

[0121]FIG. 15 illustrates the uncompensated 5.5 second DC glucoseresponse of all of the blood samples as hematocrit and temperature vary(ignoring the AC measurement data). As will be appreciated, there is awide variation in the DC current response as temperature and hematocritvary. FIG. 16 illustrates the correlation between the actual bloodglucose level of the sample versus the predicted response using Equation8. As can be seen, when the DC response is compensated for hematocritlevels using the AC response data, an overall r² value of 0.9818 isachieved with a Total Test Time of 5.5 seconds. This demonstrates theapplicability of the present invention in achieving high accuracy andfast test times with a different reagent class than was used in Examples1-3.

EXAMPLE 5 Combined AC and DC Measurement Using a 0.397 μl Sample

[0122] The measurement methods of the present invention have been foundto be useful with other test strip designs as well. Example 5 wasconducted using the test strip design illustrated in FIGS. 17A-B, andindicated generally at 1700. Referring to FIG. 17A, the test strip 1700comprises a bottom foil layer 1702 formed from an opaque piece of 350 μmthick polyester (in the preferred embodiment this is Melinex 329available from DuPont) coated with a 50 nm conductive (gold) layer (bysputtering or vapor deposition, for example). Electrodes and connectingtraces are then patterned in the conductive layer by a laser ablationprocess to form working, counter, and dose sufficiency electrodes(described in greater detail hereinbelow) as shown. The laser ablationprocess is performed by means of an excimer laser which passes through achrome-on-quartz mask. The mask pattern causes parts of the laser fieldto be reflected while allowing other parts of the field to pass through,creating a pattern on the gold which is ejected from the surface wherecontacted by the laser light.

[0123] Examples of the use of laser ablation techniques in preparingelectrodes for biosensors are described in U.S. patent application Ser.No. 09/866,030, “Biosensors with Laser Ablation Electrodes with aContinuous Coverlay Channel” filed May 25, 2001, and in U.S. patentapplication Ser. No. 09/411,940, entitled “Laser Defined Features forPatterned Laminates and Electrode,” filed Oct. 4, 1999, both disclosuresincorporated herein by reference.

[0124] The bottom foil layer 1702 is then coated in the area extendingover the electrodes with a reagent layer 1704 in the form of anextremely thin reagent film. This procedure places a stripe ofapproximately 7.2 millimeters width across the bottom foil 1702 in theregion labelled “Reagent Layer” on FIG. 17. In the present Example, thisregion is coated at a wet-coat weight of 50 grams per square meter ofcoated surface area leaving a dried reagent less than 20 μm thick. Thereagent stripe is dried conventionally with an in-line drying systemwhere the nominal air temperature is at 110° C. The rate of processingis nominally 30-38 meters per minute and depends upon the rheology ofthe reagent.

[0125] The materials are processed in continuous reels such that theelectrode pattern is orthogonal to the length of the reel, in the caseof the bottom foil 1702. Once the bottom foil 1702 has been coated withreagent, the spacer is slit and placed in a reel-to-reel process ontothe bottom foil 1702. Two spacers 1706 formed from 100 μm polyester (inthe preferred embodiment this is Melinex 329 available from DuPont)coated with 25 μm PSA (hydrophobic adhesive) on both the dorsal andventral surfaces are applied to the bottom foil layer 1702, such thatthe spacers 1706 are separated by 1.5 mm and the working, counter anddose sufficiency electrodes are centered in this gap. A top foil layer1708 formed from 100 μm polyester coated with a hydrophilic film on itsventral surface (using the process described in U.S. Pat. No. 5,997,817)is placed over the spacers 1706. In the preferred embodiment, thehydrophilic film is coated with a mixture of Vitel and Rhodapexsurfactant at a nominal thickness of 10 microns. The top foil layer 1708is laminated using a reel-to-reel process. The sensors can then beproduced from the resulting reels of material by means of slitting andcutting.

[0126] The 1.5 mm gap in the spacers 1706 therefore forms a capillaryfill space between the bottom foil layer 1702 and the top foil layer1708. The hydrophobic adhesive on the spacers 1706 prevents the testsample from flowing into the reagent under the spacers 1706, therebydefining the test chamber volume. Because the test strip 1700 is 5 mmwide and the combined height of the spacer 1706 and conductive layer is0.15 mm, the sample receiving chamber volume is

5 mm×1.5 mm×0.15 mm=1.125 μl  (Equation 9)

[0127] As shown in FIG. 17B, the distance from the sample applicationport 1710 and the dose sufficiency electrodes is 1.765 mm. The volume ofsample needed to sufficiently cover the working, counter and dosesufficiency electrodes (i.e. the minimum sample volume necessary for ameasurement) is

1.5 mm×1.765 mm×0.15 mm=0.397 μl  (Equation 10)

[0128] The reagent composition for the test strip 1700 is given inTables V and VI. TABLE V Reagent Mass Composition - Prior to Dispenseand Drying Mass for Component % w/w 1 kg solid Polyethylene oxide (300kDa) 1.0086% 10.0855 g  solid Natrosol 250M 0.3495% 3.4954 g solidCarboxymethylcellulose 7HF 0.3495% 3.4954 g solid Monobasic potassium0.9410% 9.4103 g phosphate (annhydrous) solid Dibasic potassium 1.6539%16.5394 g  phosphate (trihydrous) solid Disodium Succinate hexahydrate0.2852% 2.8516 g solid Potassium Hydroxide 0.2335% 2.3351 g solidQuinoprotein glucose 0.3321% 3.3211 g dehydrogenase (EnzC#: 1.1.99.17)solid PQQ 0.0093% 0.0925 g solid Trehalose 0.7721% 7.7210 g solidMediator 31.1144 0.6896% 6.8956 g solid Triton X-100 0.0342% 0.3419 gsolvent Water 93.7329%  937.3293 g 

[0129] TABLE VI Reagent Layer Composition - After Drying Mass perComponent % w/w Sensor* solid Polyethylene oxide (300 kDa) 15.1469% 3.7821 ug solid Natrosol 250M 5.2495% 1.3108 ug solidCarboxymethylcellulose 7HF 5.2495% 1.3108 ug solid Monobasic potassium14.1328%  3.5289 ug phosphate (annhydrous) solid Dibasic potassium24.8395%  6.2023 ug phosphate (trihydrous) solid Disodium Succinatehexahydrate 4.2827% 1.0694 ug solid Potassium Hydroxide 3.5069% 0.8757ug solid Quinoprotein glucose dehydrogenase 4.9878% 1.2454 ug (EnzC#:1.1.99.17) solid PQQ 0.1390% 0.0347 ug solid Trehalose 11.5958%  2.8954ug solid Mediator BM31.1144 10.3562%  2.5859 ug solid Triton X-1000.5135% 0.1282 ug

[0130] A protocol for the preparation of the preferred embodimentnitrosoaniline reagent is as follows:

[0131] Step 1: Prepare a buffer solution by adding 1.654 g of dibasicpotassium phosphate (trihydrous) to 31.394 g of deionized water. Mixuntil the potassium phosphate is dissolved.

[0132] Step 2: To the solution from step 1, add 0.941 g of monobasicpotassium phosphate and mix until dissolved.

[0133] Step 3: To the solution from step 2, add 0.285 g of disodiumsuccinate (hexahydrate) and mix until dissolved.

[0134] Step 4: Verify that the pH of the solution from step 3 is6.8+/−0.1. Adjustment should not be necessary.

[0135] Step 5: Prepare a 4.68 g aliquot of the solution from step 4, andto this add 229 kilounits (by DCIP assay) of the apoenzyme ofquinoprotein glucose dehydrogenase (EC#: 1.1.99.17). This isapproximately 0.3321 g. Mix, slowly, until the protein is dissolved.

[0136] Step 6: To the solution from step 5, add 9.3 milligrams of PQQand mix for no less than 2 hours to allow the PQQ and the apoenzyme toreassociate in order to provide functional enzyme.

[0137] Step 7: Prepare a solution by dissolving 0.772 g of Trehaloseinto 1.218 g of deionized water.

[0138] Step 8: After enzyme reassociation, add the solution from step 7to the solution from step 6 and continue mixing for not less than 30minutes.

[0139] Step 9: To the solution from step 4, add 0.690 g of the mediatorprecursor BM 31.1144. Mix until dissolved (this solution will have agreenish black coloration).

[0140] Step 10: Measure the pH of the solution from step 9 and adjustthe pH to a target of 7.0+/−0.1. Normally this is accomplished with1.006 g of 5N potassium hydroxide. Because the specific amount ofpotassium hydroxide may vary as needed to reach the desired pH,generally deviations in mass from the 1.006 g are made up from analiquot of 3.767 g deionized water which is also added at this step.

[0141] Step 11: Prepare a solution of Natrosol 250M (available fromAqualon), by slowly sprinkling 0.350 g over 56.191 g of deionized waterwhich is mixed (using a rotary mixer and blade impeller) at an initialrate of approximately 600 rpm in a vessel of sufficient depth such thatthe rotor blades are not exposed nor the solution running over. As theNatrosol dissolves, the mixing rate needs to be increased to a speed of1.2-1.4 krpm. Mix until the Natrosol is completely dissolved. Note thatthe resulting matrix will be extremely viscous—this is expected.

[0142] Step 12: To the solution from step 11, gradually add 0.350 g ofSodium-Carboxymethylcellulose 7HF (available from Aqualon). Mix untilthe polymer is dissolved.

[0143] Step 13: To the suspension from step 13, gradually add 1.01 g ofPolyethylene oxide of 300 kDa mean molecular weight while mixing andcontinue to mix for not less than 60 minutes before proceeding.

[0144] Step 14: Gradually add the solution from step 10 to thesuspension from step 13 while mixing.

[0145] Step 15: To the reagent from step 14, add 34.2 mg of Triton X-100(available from Roche Diagnostics) and continue mixing.

[0146] Step 16: To the reagent from step 15, add the enzyme solutionfrom step 8. Mix for no less than 30 minutes. At this point the reagentis complete. At room teperature the wet reagent mass is consideredacceptable for use for 24 hours.

[0147] The measurement results illustrated in FIG. 18 show thecorrelation coefficient r² between the DC current response and theglucose level as the Read Time varies for three combinations oftemperature and hematocrit. These results demonstrate that a robust DCresponse should be anticipated for tests as fast as 1 second. However,those skilled in the art will recognise that there are undesirablevariations in the sensor accuracy (correlation) due to the interferingeffects of temperature and hematocrit levels, suggesting that thecombined AC and DC measurement method of the present invention shouldproduce more closely correlated results.

[0148] Based upon the encouraging results obtained in FIG. 18, a furthertest was designed using the excitation signal of FIG. 19 applied to thetest strip 1700. The excitation comprised a 10 kHz AC signal applied forapproximately 1.8 seconds, a 20 kHz AC signal applied for approximately0.2 seconds, a 2 Hz AC signal applied for approximately 0.2 seconds, a 1Hz AC signal applied for approximately 0.2 seconds, and a DC signalapplied for approximately 0.5 seconds. The AC signals had an amplitudeof 12.7 mV peak, while the DC signal had an amplitude of 550 mV. TheTotal Test Time was 3.0 seconds.

[0149] A covariance study using spiked venous blood samples representingfive glucose levels (40, 120, 200, 400 and 600), five hematocrit levels(20, 30, 45, 60 and 70%) and five temperatures (12, 18, 24, 32 and 44°C.) was designed, resulting in 125 separate combinations. As in theprevious examples, the relationship between admittance, temperature andhematocrit was examined and plotted (FIG. 20 shows the admittance at 20kHz versus hematocrit as temperature varies) and it was confirmed thatthe admittance was linearly related to hematocrit in a temperaturedependent manner. An additional discovery, however, was that the phaseangle of the AC response was correlated with hematocrit in a temperatureindependent manner. The phase angle of the 20 kHz AC response is plottedversus hematocrit in FIG. 21. The results for phase angle measured at 10kHz are similar. The hematocrit of the blood sample may therefore bereliably estimated using only the phase angle information as follows:

H _(est) =c ₀ +c ₁(Φ_(10 kHz)−Φ_(20 kHz))+c₂(Φ_(2 kHz)−Φ_(1 kHz))  (Equation 11)

[0150] For the test strip used in this Example 5, the correlationbetween phase angle and hematocrit was better at higher frequencies.Because of this, the c₂ constant approaches zero and H_(est) canreliably be estimated using only the 10 kHz and 20 kHz data. Use oflower frequencies, however, allows for slight improvements in thestrip-to-strip variability of the H_(est) function. The presentinvention therefore may be used to estimate hematocrit using only ACphase angle measurements preferably made at at least one AC frequency,more preferably made at at least two AC frequencies, and most preferablymade at at least four AC frequencies.

[0151] Because the hematocrit can be determined using only the ACresponse data, and we know from FIG. 20 that admittance is linearlyrelated to hematocrit and temperature, we can now determine thetemperature of the sample under analysis using only the AC response asfollows:

T _(est) =b ₀ +b ₁(Y _(10 kHz) −Y _(20 kHz))+b ₂(Y _(2 kHz) −Y_(1 kHz))+b ₃ H _(est)  (Equation 12)

[0152] where b₀, b₁, b₂ and b₃ are constants. It will be appreciatedthat the estimation of hematocrit and temperature from the AC responsedata may be made with more or fewer frequency measurements, and atdifferent frequencies than those chosen for this example. The particularfrequencies that produce the most robust results will be determined bytest strip geometries and dimensions. The present invention thereforemay be used to estimate test sample temperature using only AC responsemeasurements preferably made at at least one AC frequency, morepreferably made at at least two AC frequencies, and most preferably madeat at least four AC frequencies.

[0153] Those skilled in the art will recognise that the directmeasurement of the temperature of the sample under test (by means of theAC response) is a great improvement over prior art methods forestimating the temperature of the sample. Typically, a thermistor isplaced in the test meter near where the test strip is inserted into themeter. Because the thermistor is measuring a temperature remote from theactual sample, it is at best only a rough approximation of the truesample temperature. Furthermore, if the sample temperature is changing(for example due to evaporation), then the thermal inertia of the testmeter and even the thermistor itself will prevent the meter-mountedthermistor from accurately reflecting the true temperature of the sampleunder test. By contrast, the temperature estimation of the presentinvention is derived from measurements made within the sample under test(i.e. within the reaction zone in which the sample under test reactswith the reagent), thereby eliminating any error introduced by thesample being remote from the measuring location. Additionally, thetemperature estimation of the present invention is made using data thatwas collected very close in time to the glucose measurement data thatwill be corrected using the temperature estimation, thereby furtherimproving accuracy. This represents a significant improvement over theprior art methods.

[0154] As a demonstration of the effectiveness of the method of thisExample 5 for correcting for the effects of interferants on the bloodglucose measurement, the uncompensated DC current response versus knownglucose concentration is plotted in FIG. 22 for all 125 combinations ofglucose, temperature and hematocrit (the AC measurements were ignoredwhen plotting this data). As will be appreciated by those skilled in theart, the data exhibits huge variation with respect to hematocrit andtemperature.

[0155] As previously discussed, the accuracy of the DC glucose responsecan be greatly improved by combining the estimated hematocrit,temperature and DC response to correct for the hematocrit andtemperature interference in the DC response as follows:

PRED=(a ₀ +hct ₁ H _(est) +hct ₂ H _(est) ² +tau ₁ T _(est) +tau ₂ T_(est))+(a ₁ DC)(1+hct ₃ H _(est) +hct ₄ H _(est) ²)(1+tau ₃ T _(est)+tau ₄ T _(est))  (Equation 13)

[0156] The constants in Equation 13 can be determined using regressionanalysis, as is known in the art. The present invention therefore allowsone to estimate hematocrit by using the AC phase angle response(Equation 11). The estimated hematocrit and the measured AC admittancecan be used to determine the estimated temperature (Equation 12).Finally, the estimated hematocrit and estimated temperature can be usedwith the measured DC response to obtain the predicted glucoseconcentration (Equation 13).

[0157] Applying the above methodology to the test data plotted in FIG.22, we obtain the predicted glucose versus DC current responseillustrated in FIG. 23. This data represents 125 covariant sampleshaving hematocrit levels ranging from 20%-70% and temperatures rangingfrom 12° C.-44° C. Even with these wide variations in interferantlevels, the measurement method of the present invention produced anoverall r² correlation of 0.9874 using a 3.0 second Total Test Time.

EXAMPLE 6 Simultaneous AC and DC Measurement Using a 0.397 μl Sample

[0158] Using the same test strip 1700 and reagent described above forExample 5, the excitation profile illustrated in FIG. 24 was utilized inorder to decrease the Total Test Time. As described above with respectto Example 5, it was determined that the phase angle at 20 kHz and at 10kHz were most closely correlated with the hematocrit estimation. It wastherefore decided to limit the AC portion of the excitation to these twofrequencies in Example 6 in order to decrease the Total Test Time. Inorder to make further reductions in Total Test Time, the 10 kHz ACexcitation was applied simultaneously with the DC signal (i.e. an ACsignal with a DC offset), the theory being that this combined mode wouldallow for the collection of simultaneous results for DC current, ACphase and AC admittance, providing the fastest possible results.Therefore, the 20 kHz signal was applied for 0.9 seconds. Thereafter,the 10 kHz and DC signals were applied simultaneously for 1.0 secondafter a 0.1 second interval.

[0159] For this Example 6, 49 spiked venous blood samples representingseven glucose levels and seven hematocrit levels were tested. Thecorrelation coefficient r² between the DC current and the bloodhematocrit was then examined at three DC measurement times: 1.1 seconds,1.5 seconds and 1.9 seconds after sample application. These correlationsare plotted versus hematocrit level in FIG. 25. All of these results arecomparable although the correlation is generally poorest at 1.1 secondsand generally best at 1.5 seconds. The minimum correlation coefficient,however, exceeds 0.99.

[0160]FIG. 26 illustrates the phase angle at 20 kHz plotted againsthematocrit levels. The correlation between these two sets of data isvery good, therefore it was decided that the 10 kHz data was unnecessaryfor estimating hematocrit. The hematocrit can therefore be estimatedsolely from the 20 kHz phase angle data as follows:

H _(est) =c ₀ +c ₁Φ_(20 kHz)  (Equation 14)

[0161]FIG. 27 illustrates the DC current response versus glucose levelfor all measured hematocrit levels as the read time is varied between1.1 seconds, 1.5 seconds and 1.9 seconds. Not surprisingly, the DCcurrent at 1.1 seconds is greater than the DC current at 1.5 seconds,which is greater than the DC current at 1.9 seconds. Those skilled inthe art will recognise that the hematocrit level has a large effect onthe DC current, particularly at high glucose concentrations.

[0162] As discussed hereinabove, the accuracy of the DC glucose responsecan be greatly improved by compensating for the interference caused byhematocrit as follows:

PRED=(a ₀ +hct ₁ H _(est) +hct ₂ H _(est) ²)+(a ₁ DC)(1+hct ₃ H _(est)+hct ₄ H _(est) ²)  (Equation 15)

[0163] Note that Equation 15 does not include temperature compensationterms since temperature variation was not included in the experiment ofthis Example 6, it can be reasonably inferred from previous examplesthat a Test term could be included using the 10 kHz and 20 kHzadmittance values in combination with the H_(est) term. Because thehematocrit can be reliably estimated using only the 20 kHz phase anglemeasurement data, the hematocrit compensated predicted glucose responsecan be determined using only this phase angle information and themeasured DC response. The compensated DC response versus glucose levelfor only the DC read at 1.1 seconds (representing a 1.1 second TotalTest Time) is illustrated in FIG. 28. The data shows an overall r²correlation of 0.9947 with a 1.1 second Total Test Time.

[0164] The same data for the 1.5 second DC read is illustrated in FIG.29, showing an overall r² correlation of 0.9932 for a 1.5 second TotalTest Time. The same data for the 1.9 second DC read is illustrated inFIG. 30, showing an overall r² correlation of 0.9922 for a 1.9 secondTotal Test Time. Surprisingly, the r² correlation actually decreasedslightly with the longer test times. Notwithstanding this, thecorrelation coefficients for all three compensated data sets—where all 7hematocrits ranging from 20% through 60% are combined—were in excess of0.99, demonstrating the applicability of the present invention to yielda blood glucose test as fast as 1.1 seconds, combined with improvedaccuracy, where the sensor requires less than 0.4 microliters of bloodin order to perform the glucose measurement test.

EXAMPLE 7 Use of AC Phase Angle to Detect an Abused Sensor

[0165] In order to provide an extra measure of quality control to theanalyte measurement process, particularly when the test system is to beused by a non-professional end user, it is desirable to detect sensors(test strips) that have been mis-dosed (double dosed, etc.), that havebeen previously used, or that have degraded enzymes (from being storedin too humid an environment, being too old, etc.). These conditions arecollectively referred to as “abused sensors.” It is desired to devise atest that will abort the analyte measurement process (or at least warnthe user that the test results may not be accurate) if an abused sensoris inserted into the test meter.

[0166] When performing a blood glucose analysis, the test meter willtypically make several successive current measurements as the bloodsample continues to react with the reagent chemistry. As is well knownin the art, this response current is known as the Cottrell current andit follows a pattern of decay as the reaction progresses. We may definea Cottrell Failsafe Ratio (CFR) as follows:

[0167] The Cottrell response of the biosensor in the Confidence systemcan be given by: $\begin{matrix}{I_{cottrell} = {\frac{{nFA}\sqrt{D}}{\sqrt{\prod\quad}}{Ct}^{\alpha}}} & \left( {{Equation}\quad 16} \right)\end{matrix}$

[0168] where:

[0169] n=electrons freed per glucose molecule

[0170] F=Faraday's Constant

[0171] A=Working electrode surface area

[0172] t=elapsed time since application of excitation

[0173] D=diffusion coefficient

[0174] C=glucose concentration

[0175] α=a cofactor-dependent constant.

[0176] All of the parameters of this equation will normally be constantfor the sensor except the glucose concentration and time. We cantherefore define a normalized Cottrell failsafe ratio (NCFR) as:$\begin{matrix}\begin{matrix}{{NCFR} = \frac{\sum\limits_{k = 1}^{m}\quad I_{k}}{{mI}_{m}}} \\{= \frac{\sum\limits_{k = 1}^{m}\quad {\frac{{nFA}\sqrt{D}}{\sqrt{\prod\quad}}{Ct}_{k}^{\alpha}}}{m\frac{{nFA}\sqrt{D}}{\sqrt{\prod\quad}}{Ct}_{m}^{\alpha}}} \\{= \frac{\sum\limits_{k = 1}^{m}\quad t_{k}^{\alpha}}{{mt}_{m}^{\alpha}}} \\{= {Constant}}\end{matrix} & \left( {{Equation}\quad 17} \right)\end{matrix}$

[0177] As the time terms in this equation are known and constant for asensor measurement, the ratio always yields a constant for Cottrellcurves with identical sample times and intervals. Therefore, the sum ofsensor currents divided by the last sensor current should yield aconstant independent of glucose concentration. This relationship is usedin the preferred embodiment to detect potentially faulty biosensorresponses.

[0178] A Current Sum Failsafe can be devised that places a check on theCottrell response of the sensor by summing all of the acquired currentsduring sensor measurement. When the final current is acquired, it ismultiplied by two constants (which may be loaded into the meter at thetime of manufacture or, more preferably, supplied to the meter with eachlot of sensors, such as by a separate code key or by information codedonto the sensor itself). These constants represent the upper and lowerthreshold for allowable NCFR values.

[0179] The two products of the constants multiplied by the final currentare compared to the sum of the biosensor currents. The sum of thecurrents should fall between the two products, thereby indicating thatthe ratio above was fulfilled, plus or minus a tolerance.

[0180] Therefore, the preferred embodiment performs the following checkwhen there is a single DC block: $\begin{matrix}{{\left( I_{m} \right)\left( C_{l} \right)} \leq {\sum\limits_{k = 1}^{m}\quad I_{k}} \leq {\left( I_{m} \right)\left( C_{u} \right)}} & \left( {{Equation}\quad 18} \right)\end{matrix}$

[0181] where

[0182] C_(u)=upper constant from the Code Key

[0183] C_(l)=lower constant from the Code Key

[0184] I_(m)=final biosensor current

[0185] Because some embodiments may contain two DC blocks in themeasurement sequence, a Modified Cottrell Failsafe Ratio (MCFR) can beformulated as: $\begin{matrix}{{MCFR} = \frac{{w_{1}{NCFR}_{1}} + {w_{2}{NCFR}_{2}}}{w_{1} + w_{2}}} & \left( {{Equation}\quad 19} \right)\end{matrix}$

[0186] Therefore, the preferred embodiment performs the following checkwhen there are two DC blocks: $\begin{matrix}{{\left( {w_{1} + w_{2}} \right)I_{m_{1}}I_{m_{2}}C_{L}} \leq \left( {{w_{1}I_{m_{2}}{\sum\limits_{k = 1}^{m_{1}}\quad I_{k}}} + {w_{2}I_{m_{1}}{\sum\limits_{k = 1}^{m_{2}}\quad I_{k}}}} \right) \leq {\left( {w_{1} + w_{2}} \right)I_{m_{1}}I_{m_{2}}C_{u}}} & \left( {{Equation}\quad 20} \right)\end{matrix}$

[0187] where

[0188] C_(u)=upper constant from the Code Key

[0189] C_(L)=lower constant from the Code Key

[0190] I_(m1), I_(m2)=final biosensor current in DC blocks 1 and 2

[0191] The NCFR (and MCFR) is correlated with hematocrit. Asdemonstrated hereinabove in Example 3, the AC phase angle is alsocorrelated with hematocrit. It follows then, that the AC phase angle andthe NCFR are correlated with one another. This relationship holds onlyif the sensor is unabused. The correlation degrades for an abusedsensor.

[0192] It is therefore possible to design an equation to analyze themeasured phase angle data to produce a failsafe calculation that willindicate if an abused sensor is being used. In the preferred embodiment,it was chosen to use the difference between the phase angles measured attwo separate frequencies in order to make the test more robust to errorscaused by parasitic resistance, etc. Applying the arctangent function todrive the two populations to different asymptotes yields the followingfailsafe equation:

FAILSAFE=1000×arctan[NCFR/(fs ₀ +fs₁(Φ_(10 kHz)−Φ_(20 kHz)))]  (Equation 21)

[0193] where

[0194] 1000=scaling factor

[0195] NCFR=Cottrell Failsafe Ratio

[0196] fs₀=linear regression intercept

[0197] fs₁=linear regression slope

[0198] Φ_(10 kHz)=phase angle at 10 kHz

[0199] Φ_(20 kHz)=phase angle at 20 kHz

[0200] Using Equation 21, the intercept term fs₀ can be chosen such thata FAILSAFE value below zero indicates an abused sensor, while a FAILSAFEvalue above zero indicates a non-abused sensor. Those skilled in the artwill recognise that the opposite result could be obtained by choosing adifferent intercept.

[0201] Use of Dose Sufficiency Electrodes

[0202] As described hereinabove, it has been recognised that accuratesample measurement requires adequate coverage of the measurementelectrodes by the sample. Various methods have been used to detect theinsufficiency of the sample volume in the prior art. For example, theAccu-Chek® Advantage® glucose test meter sold by Roche DiagnosticsCorporation of Indianapolis, Ind. warned the user of the possibleinadequacy of the sample volume if non-Cotrellian current decay wasdetected by the single pair of measurement electrodes. Users wereprompted to re-dose the test strip within a specified time allotment.

[0203] The possibility of insufficient sample size has been heightenedin recent years due to the use of capillary fill devices used inconjunction with blood lancing devices designed to minimize pain throughthe requirement of only extremely small sample volumes. If an inadequateamount of sample is drawn into the capillary fill space, then there is apossibility that the measurement electrodes will not be adequatelycovered and the measurement accuracy will be compromised. In order toovercome the problems associated with insufficient samples, variousprior art solutions have been proposed, such as placing an additionalelectrode downstream from the measurement electrodes; or a singlecounter electrode having a sub-element downstream and major elementupstream of a working electrode; or an indicator electrode arranged bothupstream and downstream from a measurement electrode (allowing one tofollow the flow progression of the sample across the working and counterelectrodes or the arrival of the sample at a distance downstream). Theproblem associated with each of these solutions is that they eachincorporate one or the other electrode of the measurement pair incommunication with either the upstream or the downstream indicatorelectrodes to assess the presence of a sufficient volume of sample toavoid biased test results.

[0204] Despite these prior art design solutions, failure modes persistwherein the devices remain prone to misinterpretation of samplesufficiency. The present inventors have determined that such erroneousconclusions are related primarily to the distances between a downstreammember of a measurement electrode pair (co-planar or opposinggeometries) and the dose detection electrode, in combination with thediversity of non-uniform flow fronts. A sample traversing the capillaryfill space having an aberrant (uneven) flow front can close the circuitbetween a measurement electrode and an indicator electrode anderroneously advise the system that sufficient sample is present to avoida biased measurement result.

[0205] Many factors employed in the composition and/or fabrication ofthe test strip capillary fill spaces influence such irregular flow frontbehavior. These factors include:

[0206] disparities between surface energies of different walls formingthe capillary fill space.

[0207] contamination of materials or finished goods in the test stripmanufacturing facility.

[0208] unintentional introduction of a contaminant from a singlecomponent making up the walls of the capillary fill space (an examplebeing a release agent (typically silicon) that is common tomanufacturing processes wherein release liners are used).

[0209] hydrophobic properties of adhesives (or contaminated adhesives)used in the lamination processes.

[0210] disparate surface roughnesses on the walls of the capillary fillspace.

[0211] dimensional aspect ratios.

[0212] contaminated mesh materials within the capillary fill space.

[0213] non-homogeneous application of surfactants onto mesh materialswithin the capillary fill space.

[0214] Another problem with prior art dose sufficiency methodologiesdetermined by the present inventors relates to the use of one or theother of the available measurement electrodes in electricalcommunication with an upstream or downstream dose detection electrode.In such arrangements, the stoichiometry of the measurement zone (thearea above or between the measurement electrodes) is perturbed duringthe dose detect/dose sufficiency test cycle prior to making ameasurement of the analyte of interest residing in the measurement zone.As sample matrices vary radically in make-up, the fill properties ofthese samples also vary, resulting in timing differences between sampletypes. Such erratic timing routines act as an additional source ofimprecision and expanded total system error metrics.

[0215] Trying to solve one or more of these obstacles typically can leadto 1) more complex manufacturing processes (additional process stepseach bringing an additional propensity for contamination); 2) additionalraw material quality control procedures; 3) more costly raw materialssuch as laminate composites having mixtures of hydrophobic andhydrophyllic resins and negatively impacting manufacturing costs; and 4)labor-intensive surfactant coatings of meshes and or capillary walls.

EXAMPLE 8 Determination of Fluid Flow Front Behavior in a Capillary FillSpace

[0216] In order to design an electrode system that will adequatelyindicate dose sufficiency in a test strip employing a capillary fillspace, an experiment was performed to examine the flow front shape atthe leading edge of the sample as it progresses through the capillaryfill space. Test fixtures comprising two sheets of clear polycarbonatesheets joined together with double-sided adhesive tape were used, wherethe capillary fill space was formed by cutting a channel in thedouble-sided tape. Use of the polycarbonate upper and lower sheetsallowed the flow fronts of the sample to be videotaped as it flowedthrough the capillary fill space.

[0217] Specifically, the test devices were laminated using laser cut 1mm thick Lexan® polycarbonate sheets (obtained from Cadillac PlasticsLtd., Westlea, Swindon SN5 7EX, United Kingdom). The top and bottompolycarbonate sheets were coupled together using double-sided adhesivetapes (#200 MP High Performance acrylic adhesive obtained from 3MCorporation, St. Paul, Minn.). The capillary channels were defined bylaser cutting the required width openings into the double-sided tape.Tape thicknesses of 0.05 μm, 0.125 μm, and 0.225 μm were used to givethe required channel heights. The dimensions of the capillary spaces ofthe test devices are tabulated in FIG. 31.

[0218] The top and bottom polycarbonate parts were laminated togetherwith the laser cut adhesive tapes using a custom-built jig to ensurereproducible fabrication. For each test device, a fluid receptor regiondefining the entrance to the capillary channel was formed by an openingpre-cut into the upper polycarbonate sheet and adhesive tape components.For each of the three channel heights, channel widths of 0.5 mm, 1.00mm, 1.5 mm, 2.00 nm, 3.00 mm, and 4.00 mm were fabricated. The capillarychannel length for all devices was 50 mm. Twenty-eight (28) of each ofthe eighteen (18) device types were constructed. The assembled deviceswere plasma treated by Weidman Plastics Technology of Dortmund, Germany.The following plasma treatment conditions were used:

[0219] Processor: Microwave plasma processor 400

[0220] Microwave Power: 600 W

[0221] Gas: O₂

[0222] Pressure: 0.39 miilibar

[0223] Gas Flow: 150 ml/min

[0224] Time: 10 minutes

[0225] Surface Energy Pre-Treatment: <38 mN/m

[0226] Surface Energy Post-Treatment: 72 mN/m

[0227] The plasma-treated devices were stored at 2-8° C. when not inuse. The devices were allowed to equilibrate to room temperature for one(1) hour minimum before use.

[0228] Each of the test devices was dosed with a fixed volume of venousblood having a hematocrit value of 45%. Flow and flow front behavior wascaptured on videotape for later analysis. It was determined that therelative dimensions of the capillary fill channel determined the flowfront behavior. Devices to the left of the dashed line in FIG. 31(devices A2, A4, B2, B4, B5, C2, C4, and C5) resulted in a convex flowfront behavior, while devices to the right of the dashed line (devicesA6, A8, A11, B6, B8, B11, C6, C8, and C11) displayed a concave flowfront behavior. Both the convex and concave flow front behaviors areschematically illustrated in FIG. 31. This data shows that the aspectratio between the height and the width of the capillary fill space is adetermining factor in whether the sample flow front is convex orconcave.

[0229] Use of Dose Sufficiency Electrodes Cont'd

[0230] The problems associated with a concave flow front in a capillaryfill space are illustrated in FIGS. 32A-C. In each of the figures, thetest strip includes a working electrode 3200, a reference electrode3202, and a downstream dose sufficiency electrode 3204 that works inconjunction with one of the measurement electrodes 3200 or 3202. Inaddition to the measurement zone stoichiometry problems associated withthe use of the dose sufficiency electrode 3204 in conjunction with oneof the measurement electrodes discussed above, FIGS. 32A-C illustratethat a sample flow front exhibiting a concave shape can also causebiased measurement results. In each drawing, the direction of sampletravel is shown by the arrow. In FIG. 32A, the portions of the sampleadjacent to the capillary walls have reached the dose sufficiencyelectrode 3204, thereby electrically completing the DC circuit betweenthis electrode and one of the measurement electrode pair that is beingmonitored by the test meter in order to make the dose sufficiencydetermination. Although the test meter will conclude that there issufficient sample to make a measurement at this time, the sample clearlyhas barely reached the reference electrode 3202 and any measurementresults obtained at this time will be highly biased.

[0231] Similarly, FIG. 32B illustrates the situation where the dosesufficiency electrode 3204 has been contacted (indicating that themeasurement should be started), but the reference electrode 3202 is onlypartially covered by the sample. Although the sample has reached thereference electrode 3202 at this time, the reference electrode 3202 isnot completely covered by sample, therefore any measurement resultsobtained at this time will be partially biased. Both of the situationsillustrated in FIGS. 32A-B will therefore indicate a false positive fordose sufficiency, thereby biasing the measurement test results. Only inthe situation illustrated in FIG. 32C, where the reference electrode3202 is completely covered by the sample, will the measurement resultsbe unbiased due to the extent of capillary fill in the measurement zone.

[0232] The present invention solves the stoichiometric problemsassociated with the prior art designs pairing the dose sufficiencyelectrode with one of the measurement electrodes when making the dosesufficiency determination. As shown in FIG. 33, the present inventioncomprehends a test strip having an independent pair of dose sufficiencyelectrodes positioned downstream from the measurement electrodes. Thetest strip is indicated generally as 3300, and includes a measurementelectrode pair consisting of a counter electrode 3302 and a workingelectrode 3304. The electrodes may be formed upon any suitable substratein a multilayer test strip configuration as is known in the art anddescribed hereinabove. The multilayer configuration of the test stripprovides for the formation of a capillary fill space 3306, also as knownin the art. Within the capillary fill space 3306, and downstream(relative to the direction of sample flow) from the measurementelectrodes 3302 and 3304 are formed a dose sufficiency working electrode3308 and a dose sufficiency counter electrode 3310, together forming adose sufficiency electrode pair.

[0233] When the test strip 3300 is inserted into the test meter, thetest meter will continuously check for a conduction path between thedose sufficiency electrodes 3308 and 3310 in order to determine when thesample has migrated to this region of the capillary fill space. Once thesample has reached this level, the test meter may be programmed toconclude that the measurement electrodes are covered with sample and thesample measurement sequence may be begun. It will be appreciated that,unlike as required with prior art designs, no voltage or current need beapplied to either of the measurement electrodes 3302 and 3304 during thedose sufficiency test using the test strip design of FIG. 33. Thus thestoichiometry of the measurement zone is not perturbed during the dosesufficiency test cycle prior to making a measurement of the analyte ofinterest residing in the measurement zone. This represents a significantimprovement over the dose sufficiency test methodologies of the priorart.

[0234] The test strip 3300 is also desirable for judging dosesufficiency when the capillary fill space is designed to produce samplesthat exhibit a convex flow front while filling the capillary fill space3306, as illustrated in FIG. 34A. As can be seen, the measurement zoneabove the measurement electrodes 3302 and 3304 is covered with samplewhen the convex flow front reaches the dose sufficiency electrode pair3308,3310. The test strip design 3300 may not, however, produce idealresults if the capillary fill space 3306 allows the sample to exhibit aconcave flow front while filling, as shown in FIG. 34B. As can be seen,the peripheral edges of the concave flow front reach the dosesufficiency electrodes 3308,3310 before the measurement zone has beencompletely covered with sample. With DC or low frequency excitation(discussed in greater detail hereinbelow), the dose sufficiencyelectrodes 3308,3310 will indicate sample sufficiency as soon as theyare both touched by the edges of the flow front. Therefore, the dosesufficiency electrode design shown in the test strip of FIG. 33 worksbest when the sample filling the capillary space 3306 exhibits a convexflow front.

[0235] It will be appreciated that the dose sufficiency electrodes3308,3310 have their longest axis within the capillary fill space 3306oriented perpendicular to the longitudinal axis of the capillary fillspace 3306. Such electrodes are referred to herein as “perpendiculardose sufficiency electrodes.” An alternative dose sufficiency electrodearrangement is illustrated in FIGS. 35A-B. As shown in FIG. 35A, thepresent invention also comprehends a test strip having an independentpair of dose sufficiency electrodes positioned downstream from themeasurement electrodes, where the dose sufficiency electrodes have theirlongest axis within the capillary fill space oriented parallel to thelongitudinal axis of the capillary fill space. Such electrodes arereferred to herein as “parallel dose sufficiency electrodes.” The teststrip in FIG. 35 is indicated generally as 3500, and includes ameasurement electrode pair consisting of a counter electrode 3502 and aworking electrode 3504. The electrodes may be formed upon any suitablesubstrate in a multilayer test strip configuration as is known in theart and described hereinabove. The multilayer configuration of the teststrip provides for the formation of a capillary fill space 3506, also asknown in the art. Within the capillary fill space 3506, and downstream(relative to the direction of sample flow) from the measurementelectrodes 3502 and 3504 are formed a dose sufficiency working electrode3508 and a dose sufficiency counter electrode 3510, together forming aparallel dose sufficiency electrode pair.

[0236] When the test strip 3500 is inserted into the test meter, thetest meter will continuously check for a conduction path between thedose sufficiency electrodes 3508 and 3510 in order to determine when thesample has migrated to this region of the capillary fill space. Once thesample has reached this level, the test meter may be programmed toconclude that the measurement electrodes are covered with sample and thesample measurement sequence may be begun. It will be appreciated that,as with the test strip 3300 (and unlike as required with prior artdesigns), no voltage or current need be applied to either of themeasurement electrodes 3502 and 3504 during the dose sufficiency testusing the test strip design of FIG. 35. Thus the stoichiometry of themeasurement zone is not perturbed during the dose sufficiency test cycleprior to making a measurement of the analyte of interest residing in themeasurement zone. This represents a significant improvement over thedose sufficiency test methodologies of the prior art.

[0237] A further improved operation is realized with the parallel dosesufficiency electrodes of the test strip 3500 when the dose sufficiencyelectrodes are energized with a relatively high frequency AC excitationsignal. When a relatively high frequency AC signal is used as the dosesufficiency excitation signal, the dose sufficiency electrodes 3508,3510display significant edge effects, wherein the excitation signaltraverses the gap between the electrodes only when the electrode edgesalong the gap are covered with the sample fluid. The test strip 3500 isillustrated in enlarged size in FIG. 36 (with only the electrodeportions lying within the capillary fill space 3506 and thestrip-to-meter electrode contact pads visible). When one of the pair ofdose sufficiency electrodes 3508,3510 is excited with an AC signal, themajority of the signal travels from one electrode edge to the edge ofthe other electrode (when the edges are covered with sample), ratherthan from the upper flat surface of one electrode to the upper flatsurface of the other electrode. These paths of edge-to-edge electricalcommunication are illustrated schematically as the electric field lines3602 in FIG. 36.

[0238] Higher AC frequencies produce the best edge-only sensitivity fromthe dose sufficiency electrodes. In the preferred embodiment, a 9mV_(rms) (+/−12.7 mV peak-to-peak) excitation signal of 10 kHz is usedto excite one of the dose sufficiency electrodes. The gap width GWbetween the edges of the dose sufficiency electrodes 3508,3510 ispreferably 100-300 μm, more preferably 150-260 μm, and most preferably255 μm. A smaller gap width GW increases the amount of signaltransmitted between dose sufficiency electrodes whose edges are at leastpartially covered by sample; however, the capacitance of the signaltransmission path increases with decreasing gap width GW.

[0239] An advantage of the parallel dose sufficiency electrode design ofFIGS. 35 and 36, when used with AC excitation, is that there issubstantially no electrical communication between the electrodes untilthe sample covers at least a portion of the edges along the electrodegap. Therefore, a sample exhibiting the concave flow front of FIG. 35A,where the illustrated sample is touching both of the dose sufficiencyelectrodes 3508,3510 but is not touching the electrode edges along thegap, will not produce any significant electrical communication betweenthe dose sufficiency electrodes. The test meter will therefore not forma conclusion of dose sufficiency until the sample has actually bridgedthe dose sufficiency electrodes between the electrode edges along thegap. This will happen only after the rear-most portion of the concaveflow front has reached the dose sufficiency electrodes 3508,3510, atwhich point the sample has completely covered the measurement zone overthe measurement electrodes. As can be seen in FIG. 35B, convex sampleflow fronts will activate the dose sufficiency electrodes 3508,3510 assoon as the flow front reaches the dose sufficiency electrodes (at whichpoint the sample has completely covered the measurement zone over themeasurement electrodes).

[0240] Another advantage to the parallel dose sufficiency electrodesillustrated in FIGS. 35 and 36 is that the amount of signal transmittedbetween the electrodes is proportional to the amount of the gap edgesthat is covered by the sample. By employing an appropriate thresholdvalue in the test meter, a conclusion of dose sufficiency can thereforebe withheld until the sample has covered a predetermined portion of thedose sufficiency electrode gap edge. Furthermore, an analysis of thedose sufficiency signal will allow the test meter to record thepercentage of fill of the capillary fill space for each measurement madeby the test meter, if desired.

[0241] While the electrode geometry itself demonstrates an advantageover previous embodiments in terms of detecting an adequate sample,particularly in the case of a convex flow front, it was found thatfurther improvement is achieved in the use of AC responses over DCresponses for sample detection. DC responses have the problems of beingsensitive to variations in, for example, temperature, hematocrit and theanalyte (glucose for example). AC responses at sufficiently highfrequency can be made robust to the variation in the analyteconcentration. Further, the AC response generated at sufficiently highfrequencies in such capillary fill devices is primarily limited by theamount of the parallel gap between the electrode edges which is filledby the sample. Thus, for a convex flow front, little or no AC response(in this case admittance) is perceived until the trough of the flowfront actually intrudes within the parallel edges of the samplesufficiency electrodes. Further, by means of threshold calibration, thesensor can be made more or less sensitive as is deemed advantageous,with a higher threshold for admittance requiring more of the parallelgap to be filled before test initiation.

[0242] A further limitation of existing devices is the inability of theelectrode geometry to discern the amount of time needed to fill thecapillary space of the sensor. This limitation is caused by havinginterdependence of the dose sufficiency electrode and the measurementelectrodes. This is a further advantage of independent dose sufficiencyelectrodes. In the preferred embodiment a signal is first applied acrossthe measurement electrodes prior to dosing. When a response is observed,the potential is immediately switched off and a second signal is appliedacross the dose sufficiency electrodes during which time the system bothlooks for a response to the signal (indicating electrode coverage) andmarks the duration between the first event (when a response is observedat the measurement electrodes) and the second event (when a response isobserved at the dose sufficiency electrodes). In cases where very longintervals may lead to erroneous results, it is possible to establish athreshold within which acceptable results may be obtained and outside ofwhich a failsafe is triggered, preventing a response or at a minimumwarning the user of potential inaccuracy. The amount of time lag betweendosing and detection of a sufficient sample that is considered allowableis dependent upon the particular sensor design and chemistry.Alternatively, an independent pair of dose detection electrodes (notshown) may be added upstream from the measurement electrodes in order todetect when the sample is first applied to the sensor.

[0243] While a DC signal could be used for detection in either or bothof the above events, the preferred embodiment uses an AC signal atsufficiently high frequency to avoid unnecessarily perturbing theelectrochemical response at the measurement electrodes and to providerobust detection with respect to flow front irregularities.

[0244] All publications, prior applications, and other documents citedherein are hereby incorporated by reference in their entirety as if eachhad been individually incorporated by reference and fully set forth.

[0245] While the invention has been illustrated and described in detailin the drawings and foregoing description, the description is to beconsidered as illustrative and not restrictive in character. Only thepreferred embodiment, and certain other embodiments deemed helpful infurther explaining how to make or use the preferred embodiment, havebeen shown. All changes and modifications that come within the spirit ofthe invention are desired to be protected.

What is claimed is:
 1. A method for determining a glucose concentrationof a blood sample, comprising the steps of: a) applying a signal havingan AC component to the blood sample; b) measuring an AC phase angleresponse to the signal; and c) determining the glucose concentrationusing the AC phase angle response.
 2. The method of claim 1, whereinstep (c) comprises determining an effective phase angle, which isproportional to the glucose concentration, using the AC phase angleresponse.
 3. The method of claim 2, wherein step (c) comprisesdetermining the effective phase angle using P _(eff)=(Φ/Γ)^(−γ) Where:P_(eff) is the effective phase angle, Φ is the AC phase angle response,and ⊖ and γ are constants.
 4. The method of claim 1, wherein step (a)comprises applying a signal having a frequency of 2 kHz or below.
 5. Themethod of claim 1, wherein step (a) comprises applying a signal having afrequency of 1 kHz or below.
 6. The method of claim 1, wherein step (a)comprises applying a signal having a frequency of 200 Hz or below. 7.The method of claim 2, wherein step (c) further comprises determiningthe glucose concentration using PRED=(a ₀ +hct ₁ H _(est) +hct ₂ H_(est) ² +tau ₁ dT+tau ₂ dT ²)+(a ₁ P _(eff))(1+hct ₃ H _(est) +hct ₄ H_(est) ²)(1+tau ₃ dT+tau ₄ dT ²) Where: PRED is the glucoseconcentration, P_(eff) is the effective phase angle, a₀, a₁, hct, hct₂,hct₃, hct₄, tau₁, tau₂, tau₃ and tau₄ are constants, H_(est) is ahematocrit value of the blood sample, and dT is the temperature
 8. Themethod of claim 3, wherein step (a) comprises applying a signal having afrequency of 2 kHz or below.
 9. The method of claim 1, wherein thesignal is an AC signal.
 10. The method of claim 1, wherein the ACcomponent of the signal has a frequency not less than 1 Hz and notgreater than 20 kHz.
 11. A method of determining a glucose concentrationof a biological fluid sample, comprising: (a) applying a signal havingan AC component to the sample; (b) measuring an AC phase angle responseto the signal; and (c) determining the glucose concentration based uponthe AC phase angle response and a predetermined correlation between theAC phase angle response and the glucose concentration.
 12. The method ofclaim 11, wherein the biological fluid is blood.
 13. The method of claim12, wherein step (c) comprises determining an effective phase angle,which is proportional to the glucose concentration, using the AC phaseangle response.
 14. The method of claim 13, wherein step (c) comprisesdetermining the effective phase angle using P _(eff)=(Φ/⊖)^(−γ) Where:P_(eff) is the effective phase angle, Φ is the AC phase angle response,and ⊖ and γ are constants.
 15. The method of claim 11, wherein step (a)comprises applying a signal having a frequency of 2 kHz or below. 16.The method of claim 11, wherein step (a) comprises applying a signalhaving a frequency of 1 kHz or below.
 17. The method of claim 11,wherein step (a) comprises applying a signal having a frequency of 200Hz or below.
 18. The method of claim 13, wherein step (c) furthercomprises determining the glucose concentration using PRED=(a ₀ +hct ₁ H_(est) +hct ₂ H _(est) ² +tau ₁ dT+tau ₂ dT ²)+(a ₁ P _(eff))(1+hct ₃ H_(est) +hct ₄ H _(est) ²)(1+tau ₃ dT+tau ₄ dT ²) Where: PRED is theglucose concentration, P_(eff) is the effective phase angle, a₀, a₁,hct, hct₂, hct₃, hct₄, tau₁, tau₂, tau₃ and tau₄ are constants, H_(est)is a hematocrit value of the blood sample, and dT is the temperature 19.The method of claim 14, wherein step (a) comprises applying a signalhaving a frequency of 2 kHz or below.
 20. The method of claim 11,wherein the signal is an AC signal.
 21. The method of claim 11, whereinthe AC component of the signal has a frequency not less than 1 Hz andnot greater than 20 kHz.
 22. A method of determining a glucoseconcentration of a test sample comprising: (a) applying a signal havingan AC component to the sample; (b) measuring an AC phase angle responseto the signal; and (c) determining the glucose concentration using thefirst AC phase angle response and a predetermined compensation factor.23. The method of claim 22, wherein the predetermined compensationfactor accounts for a test sample temperature.
 24. The method of claim22, wherein the test sample is blood.
 25. The method of claim 24,wherein the predetermined compensation factor accounts for a test samplehematocrit value.
 26. The method of claim 24, wherein the predeterminedcompensation factor accounts for a test sample temperature and a testsample hematocrit value.
 27. The method of claim 24, wherein step (c)comprises determining an effective phase angle, which is proportional tothe glucose concentration, using the AC phase angle response.
 28. Themethod of claim 27, wherein step (c) comprises determining the effectivephase angle using P _(eff)=(Φ/⊖)^(−γ) Where: P_(eff) is the effectivephase angle, Φ is the AC phase angle response, and ⊖ and γ areconstants.
 29. The method of claim 22, wherein step (a) comprisesapplying a signal having a frequency of 2 kHz or below.
 30. The methodof claim 22, wherein step (a) comprises applying a signal having afrequency of 1 kHz or below.
 31. The method of claim 22, wherein step(a) comprises applying a signal having a frequency of 200 Hz or below.32. The method of claim 27, wherein step (c) further comprisesdetermining the glucose concentration using PRED=(a ₀ +hct ₁ H _(est)+hct ₂ H _(est) ² +tau ₁ dT+tau ₂ dT ²)+(a ₁ P _(eff))(1+hct ₃ H _(est)+hct ₄ H _(est) ²)(1+tau ₃ dT+tau ₄ dT ²) Where: PRED is the glucoseconcentration, P_(eff) is the effective phase angle, a₀, a₁, hct, hct₂,hct₃, hct₄, tau₁, tau₂, tau₃ and tau₄ are constants, H_(est) is ahematocrit value of the blood sample, and dT is the temperature
 33. Themethod of claim 28, wherein step (a) comprises applying a signal havinga frequency of 2 kHz or below.
 34. The method of claim 22, wherein thesignal is an AC signal.
 35. The method of claim 22, wherein the ACcomponent of the signal has a frequency not less than 1 Hz and notgreater than 20 kHz.
 36. A method for determining a hematocrit value ofa blood sample, comprising the steps of: a) applying at least one signalhaving an AC component to the blood sample; b) measuring at least one ACphase angle response to respective ones of the at least one signal; andc) determining the hematocrit value using the at least one AC phaseangle response.
 37. The method of claim 36, wherein said at least onesignal comprises at least two frequencies.
 38. The method of claim 36,wherein said at least one signal comprises at least four frequencies.39. The method of claim 37, wherein said at least two frequencies areapplied at least partially simultaneously.
 40. The method of claim 38,wherein said at least four frequencies are applied at least partiallysimultaneously.
 41. The method of claim 36, wherein said at least onesignal comprises n signals, and wherein step (c) comprises determiningthe hematocrit value using H _(est) =c ₀ +c ₁Φ₁ . . . c _(n)Φ_(n) Where:H_(est) is the hematocrit value, c₀, c₁ . . . c_(n) are constants, andΦ₁ . . . Φ_(n) are respective AC phase angle responses to each of the nsignals.
 42. A method for determining a hematocrit value of a bloodsample, comprising the steps of: (a) applying a first signal having anAC component to the blood sample, the first signal having a firstfrequency; (b) measuring a first AC phase angle response to the firstsignal; (c) applying a second signal having an AC component to the bloodsample, the second signal having a second frequency; (d) measuring asecond AC phase angle response to the second signal; and (e) determiningthe hematocrit value based at least in part upon the first phase angleresponse and the second phase angle response.
 43. The method of claim42, further comprising the steps of: (f) applying a third signal havingan AC component to the blood sample, the third signal having a thirdfrequency; (g) measuring a third AC phase angle response to the thirdsignal; (h) applying a fourth signal having an AC component to the bloodsample, the fourth signal having a fourth frequency; and (i) measuring afourth AC phase angle response to the fourth signal; (j) wherein thedetermining the hematocrit value is further based upon the third phaseangle response and the fourth phase angle response.
 44. The method ofclaim 42 wherein the first frequency is about 10 kHz and the secondfrequency is about 20 kHz.
 45. The method of claim 43 wherein the thirdfrequency is about 2 kHz and the fourth frequency is about 1 kHz. 46.The method of claim 42, wherein the first frequency and the secondfrequency are applied at least partially simultaneously.
 47. The methodof claim 43, wherein the first frequency, the second frequency, thethird frequency and the fourth frequency are applied at least partiallysimultaneously.
 48. The method of claim 43, wherein step (j) comprisesdetermining the hematocrit value using H _(est) =c ₀ +c ₁Φ₁ +c ₂Φ₂ +c₃Φ₃ +c ₄Φ₄ Where: H_(est) is the hematocrit value, c₀, c₁, c₂, c₃, c₄are constants, and Φ₁,Φ₂,Φ₃,Φ₄ are respective AC phase angle responsesto the first, second, third and fourth signals.